1. Field of the Invention
The present invention is directed generally to processes for improving human tissue plasminogen activator (t-PA) protein and, in particular, to processes for improving the pharmacokinetic characteristics of t-PA.
2. Description of the Related Art
Human tissue plasminogen activator, t-PA, is an extremely important new biological pharmaceutical agent shown to have great promise in the treatment of vascular disease due to its high specificity and potent ability to dissolve blood clots in vivo. Accordingly, t-PA has been hailed by medical science as one of the most impressive new agents of recent history for the treatment of vascular disease, and in particular, heart disease. For these and other reasons, t-PA will likely revolutionize the clinical management of serious vascular disease.
Human t-PA protein, as well as the underlying gene sequences which code for it, has been the subject of numerous scientific disclosures over the previous few years. For example, the structure of t-PA protein, as well as its isolation from natural sources, has been described by Rijken et al., (1981), J. Biol. Chem., 256:7035. Moreover, a patent and various patent applications have been published detailing the isolation of natural t-PA from both natural and recombinant sources (see, e.g., UK Patent 2,119,804; European Patent Application Publication No. 041766; and European Patent Application Publication No. 093619). Based on such disclosures, it is now clear that natural t-PA, whether naturally isolated or a recombinant species thereof, typically includes 5 domains which have been defined with reference to homologous or similar structures identified in various other proteins. These domains have been designated as the finger (F), growth factor (G), kringle 1 (K1), kringle 2 (K2), and protease (P) regions and are situated contiguously in the N-terminus to C-terminus direction of the protein backbone structure.
In spite of the profound advantages identified with natural human t-PA as a clot dissolving pharmaceutical agent, certain drawbacks are associated with its use under various circumstances. For example, natural t-PA has an extremely short plasma half-life, typically about 6 minutes or so, when administered to patients in therapeutically effective amounts. Moreover, in terms of clearance rate, another important pharmacokinetic indicator, natural t-PA typically exhibits an extremely high clearance rate of about 7 to 8.5 ml/min/kg. Short half-lives and high clearance rates are desirable under certain circumstances, for example, when acute aggressive therapy of a life threatening disease such as myocardial infarction or pulmonary embolism is undertaken. In this high risk situation, patients may be treated who have significant or unrecognized potential for uncontrolled bleeding. If such bleeding would occur, drug administration could be stopped and the causative t-PA levels would be rapidly depleted by high clearance rates.
However, in other circumstances, for example, in the treatment of myocardial infarction following reperfusion, the desired therapeutic regimen is less aggressive and of extended duration (4 to 12 hours). A long half-life form of t-PA can be perceived as a more desirable, efficient and convenient treatment in patients who are not in life-threatening situations. Moreover, a longer half-life t-PA would be desirable as an agent for bolus administration, for example, by ambulance technicians, where infusion capability is generally not available, it would be much more desirable to employ t-PA-like agents having greater half-lives and/or lower clearance rates.
Accordingly, there is currently a need to identify improved processes and associated embodiments for preparing t-PA variants having improved pharmacokinetic parameters yet which retain high clot lysis activity in vivo. Such would provide medical science important new alternatives to the treatment of cardiovascular disease and in treatment of numerous other medical conditions which arise out of thromboembolic occlusion of blood vessels.